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Studies on biosynthesis, biodegradation, and regulation mechanism γ-polyglutamic acid in Bacillus subtilis

枯草芽孢杆菌γ-聚谷氨酸生物合成、生物降解及调控机制研究

基本信息

批准号：
15580058
负责人：
TAHARA Yasutaka
金额：
$ 2.43万
依托单位：
Shizuoka university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Among PGA operon (ywsC-ywtABC) involved in γ-polyglutamic acid biosynthesis in Bacillus subtilis, no production of PGA was observed in ΔywsC and ΔywtA strains, but ΔywtB strain producing a small amount of PGA with 100% L-Glu. However, the ΔywtB revertant produced PGA with 70 : 30 in ratio of D, L-Glu, which is same to that of the wild-type. There are two glutamate racemases, GIr and YrpC, in B. subtilis, of which the genes were disrupted in an attempt to make clear determination of D, L-glutamate in PGA. ΔywtB-ΔgIr-ΔyrpC strain produced PGA with only L-Glu as well as that of the wild type. These results, together with the fact that the purified YwsC is able to biosynthesizes ^<14>C-PGA only from ^<14>C-L-glutamate under presence of ATP and Mg^<2+>, suggest that YwtB should possess the activity of glutamate racemase for producing PGA heterogeneous with D, L-glutamate.The structure of γ-polyglutamate-hydrolyzed product (F2) having about 2 kDa in molecular mass with D-glutamate and L-glutamate in an 80 : 20 ratio released by YwtD of B. subtilis was analyzed using γ-glutamyltranspeptidase, rat γ-glutamyl hydrolase and carboxypeptidase G. The analysis of the digested products showed the F2 product is an optically heterogeneous polymer consisting of D-γ-glutamate and L-γ-glutamate peptide units with D-glutamate in both sides, suggesting that the YwtD should be an unique endopeptidase that cleaves γ-glutamyl bond between D-and D-glutamate recognizing adjacent L-glutamate in the N-terminal side.

枯草芽孢杆菌参与γ-聚谷氨酸生物合成的PGA操纵子（ywsC-ywtABC）中，ΔywsC和ΔywtA菌株未观察到PGA的产生，而ΔywtB菌株以100% L-Glu产生少量PGA。而ΔywtB逆转录酶产生的PGA与野生型的D、L-Glu的比例为70:30，与野生型相同。在枯草芽孢杆菌中存在两个谷氨酸外消旋酶GIr和YrpC，为了明确PGA中D， l -谷氨酸的含量，对其基因进行了破坏。ΔywtB-ΔgIr-ΔyrpC菌株与野生型一样，只含L-Glu产生PGA。这些结果，再加上纯化的YwsC在ATP和Mg^<2+>存在下仅能从^<14> c - l -谷氨酸合成^<14>C-PGA，表明YwtB应该具有谷氨酸消旋酶的活性，可以产生与D， l -谷氨酸异质的PGA。γ-聚谷氨酸水解产物（F2）与d -谷氨酸和l -谷氨酸的分子质量约为2 kDa。利用γ-谷氨酰转肽酶、大鼠γ-谷氨酰水解酶和羧肽酶g对枯草芽孢杆菌YwtD释放的20比值进行了分析。分析结果表明，F2产物是一种光学非均相聚合物，由D-γ-谷氨酸和L-γ-谷氨酸肽单元组成，两侧有D-谷氨酸，表明YwtD应该是一种独特的内肽酶，它可以在D-谷氨酸和D-谷氨酸之间切割γ-谷氨酸键，识别n端相邻的L-谷氨酸。